Genetic testing has been central to various fields, such as drug development, forensic medicine, clinical tests, and identification of agricultural products or pathogenic microorganisms. Genetic testing serves as a ubiquitous technique for, for example, disease diagnosis and prognosis, marker selection, safety evaluation of food products and environments, and identification of crime scene evidence. Genetic testing is well known for being used in tests for confirming infectious diseases, such as foot-and-mouth disease and new pandemic influenza, which became issues last year. In 2007, the Health Insurance Bureau of the Ministry of Health, Labour and Welfare of Japan approved oncogene testing to be covered by insurance. Since then, clinical test-related companies have announced the commercialization of apparatus or kits for genetic testing, and genetic testing is now gaining momentum in medical treatment as well.
One of the most powerful and basic techniques for detecting a small amount of nucleic acid, i.e., a gene, in a highly sensitive manner is to exponentially replicate some or all of the nucleic acid sequence, and analyze the amplification product.
The polymerase chain reaction (PCR) is a powerful technique used to selectively amplify a certain specific region of DNA. PCR can generate millions of copies of DNA fragments of a target DNA sequence from a single template DNA. PCR is performed by repeating a three-phase temperature condition, which is called a thermal cycle. Specifically, the following individual reactions are successively repeated: denaturation of DNA into single-stranded DNA; annealing of primers to the denatured single-stranded DNA; and extension of the primers by a thermostable DNA polymerase. This cycle is repeated until a number of copies sufficient for analysis is obtained. In principle, each cycle of PCR can double the number of copies. In practice, as thermal cycling continues, the buildup of amplified DNA products eventually ceases, since the reaction reagent concentration decreases to a level lower than that required for the reaction to proceed. For the general details of PCR, see “Clinical Applications of PCR,” Dennis Lo (ed.), Humana Press (Totowa, N.J.) (1998), and “PCR Protocols: A Guide to Methods and Applications,” M. A. Innis et al. (ed.), Academic Press Inc. (San Diego, Calif.) (1990).
The PCR method is a powerful technique used to exponentially amplify genes by thermal cycling. However, in a generally used thermal cycling device used in PCR, the temperature control is slow due to the huge thermal capacity of the aluminum block heater; thus, the PCR procedure requires 1 to 2 hours for 30 to 40 cycles. Even when the latest genetic testing device is used, the analysis requires a total of several hours. Therefore, speeding up the PCR procedure has been a major object since this technique was introduced.
To achieve the above object, a microfluidic device related to DNA amplification by PCR has also been developed. Thermal cycling of the sample is usually accomplished by one of three methods.
In the first method, the sample solution is loaded into the device, and the temperature cycling is performed over time while the solution is maintained at the same position. This is much like a conventional PCR instrument (Non-patent Documents 1 and 2, and Patent Document 1). Although the purpose of this method is to speed up thermal cycling by reducing the sample amount to reduce the thermal capacity, the reduction in the thermal capacity of the heater or chamber itself is limited, and at least about 30 seconds is required per cycle to sufficiently perform the amplification reaction; therefore, even with the use of the highest-speed device, 15 minutes or longer must be spent to complete the PCR reaction.
In the second method, a plurality of temperature zones spatially apart from each other are connected through a micro-flow channel, and the sample solution is heated while moving back and forth from one zone to another in the same flow channel, in such a manner that the sample stays in each temperature zone for a predetermined time. This method is excellent in that thermal cycling can be performed by arbitrarily setting the time for each temperature zone. However, a number of integrated valves and pumps are used to introduce the sample and pump it through the temperature zones in a rotary fashion; thus, downsizing the device is difficult (Patent Document 3).
In the third method, called continuous-flow PCR, the sample solution is continuously fed in, without being stopped, to move through a plurality of temperature zones spatially separated from each other via a micro-flow channel, similar to the second method. Of the continuous-flow PCR methods, one that is attracting attention is a system for rapidly controlling the sample temperature by allowing the sample to flow through a serpentine channel on three heaters, each having a certain controlled temperature (Non-patent Document 3). In this system, it is not necessary to change the temperatures of external devices such as containers and heaters. Therefore, in theory, this system is expected to achieve the temperature control at the highest speed. In view of this, developments have been made to realize commercialization of this system. However, this system has not yet been put to practical use, as it suffers from problems such as the flow frequently being stopped due to air bubbles randomly formed at a heating zone. Specifically, in continuous-flow PCR, a PCR sample is continuously introduced so as to fill the entire micro-flow channel through 2 to 3 individual temperature zones. However, such a continuous flow requires a large amount of PCR sample, and complicated controls. Further, air bubbles are easily formed at the denaturation temperature zone at 95° C., frequently causing the disturbance and stopping of the flow. Moreover, the sample solution passes through several meters of a microtube or micro-flow channel to repeatedly move through each temperature zone about 30 to 40 times; thus, the fluid resistance becomes large as the flow speed becomes slow, preventing efficient and rapid temperature control from being achieved. As a result, about 1 hour is required to complete continuous-flow PCR; even with the use of a high-speed system, 15 minutes or more is required.
The market for genetic testing using PCR/real-time PCR devices has been growing stably. In particular, genetic testing for infectious diseases, such as viral hepatitis, sexually transmitted diseases, and influenza, has become more prevalent domestically. Further, the usefulness of genetic testing in cancer treatment has become clear. For example, an EGFR gene mutation can be used, for example, as an indication for application of the anticancer drug Iressa. Based on this fact, genetic testing in relation to EGFR gene, K-RAS gene, EWS/Fli-1 gene, TLS-CHOP gene, SVT-SSX gene, and c-kit gene in lung cancer, pancreatic cancer, and the like, has recently become insurance-covered.
Under current conditions, a sample is transported to a laboratory or an analytical center to perform genetic testing. However, if a high-speed genetic testing system, which can be quickly used on-site, is available, a plan of treatment or countermeasure can be determined instantly. Such a system can thus be considered a ground-breaking technique to use in place of currently available genetic testing devices. In particular, to prevent pandemics such as foot-and-mouth disease and highly pathogenic avian influenza from occurring, important factors are a quick and accurate decision on-site, as well as prevention of a secondary infection associated with migration. Therefore, there is a great need for a high-speed genetic testing system. In particular, a high-speed and simple genetic testing technique that can be performed at a low cost is required to achieve realization of services for promptly performing genetic testing at a clinical location or at the location of an infectious disease onset.
However, a quick and simple PCR that can be performed on-site is not available with current technology, and a method for ultra-rapidly performing amplification has been in demand.